Poster Presentation Australasian Society for Immunology Annual Scientific Meeting 2014

Expansion of influenza haemagglutinin-specific CD4+ regulatory T cells from peripheral blood of a HLA-DRB1*04:01+ healthy donor (#319)

Jennifer R Tyler 1 , Hanno Nel 1 , Soi Cheng Law 1 , Hugh Reid 2 , Jamie Rossjohn 2 , Ranjeny Thomas 1
  1. The University of Queensland Diamantina Institute, Translational Research Institute, Princess Alexandra Hospital, University of Queensland Diamantina Institute, Brisbane, Queensland, Australia
  2. Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia

Using peptide-HLA tetramer technology, antigen-specific T cells can be identified and isolated in individuals carrying specific HLA types.  Using influenza haemagglutinin (HA)306-318-HLA-DRB1*04:01 tetramers, we previously  demonstrated antigen-specific CD4+ T cells amongst peripheral blood mononuclear cells (PBMC) of healthy HLA-DRB1*0401+ human donors, of which 50-90% had a phenotype of CD25+Foxp3+CD45RO+ or CD45RO- regulatory T (Treg) cells.  The aim of this study was to clone and characterize the function of HA-specific CD4+ T cells using single-cell-sorting from PBMC prepared from a healthy HLA-DRB1*0401+ human donor. Single pHLA-tetramer+ cells were sorted into wells with allogeneic feeder cells, stimulated with mitogen and expanded with IL-2. Of the 32 clones which were generated, 19 responded specifically to HA peptide restimulation. Although slow-growing in the presence of 50 IU/ml IL-2, these clones were easily expanded in presence of 300 IU/ml of IL-2 and anti-CD3/CD28-coated beads.  By flow cytometry, 3/4 expanded pHLA-tetramer+ clones were CD4+ CD25+ FOXP3high CD45RA-, characteristic of activated Treg cells.  We examined their suppressive function by co-culture of equal numbers of autologous conventional T cells and cloned cells for six days, in presence of anti-CD3/CD28-coated beads.  In response to this polyclonal stimulus, both pHLA-tetramer+ and pHLA-tetramer- Foxp3+ clones suppressed conventional T cell proliferation. These preliminary data indicate that CD4+ clones with regulatory function can be expanded from healthy donors based on single-cell sorting of pHLA tetramer+ T cells directly ex vivo.